8-azaprostaglandin analogs as agents for lowering intraocular pressure

ABSTRACT

The present invention provides a method of treating ocular hypertension or glaucoma which comprises administering to an animal having ocular hypertension or glaucoma therapeutically effective amount of a compound represented by the general formula I;wherein X, Y, Z, D and R3 are as defined in the specification.

FIELD OF THE INVENTION

The present invention relates 8-Azaprostaglandin analogues as potent ocular hypotensives that are particularly suited for the management of glaucoma.

BACKGROUND OF THE INVENTION Description of Related Art

Ocular hypotensive agents are useful in the treatment of a number of various ocular hypertensive conditions, such as post-surgical and post-laser trabeculectomy ocular hypertensive episodes, glaucoma, and as presurgical adjuncts.

Glaucoma is a disease of the eye characterized by increased intraocular pressure. On the basis of its etiology, glaucoma has been classified as primary or secondary. For example, primary glaucoma in adults (congenital glaucoma) may be either open-angle or acute or chronic angle-closure. Secondary glaucoma results from pre-existing ocular diseases such as uveitis, intraocular tumor or an enlarged cataract.

The underlying causes of primary glaucoma are not yet known. The increased intraocular tension is due to the obstruction of aqueous humor outflow. In chronic open-angle glaucoma, the anterior chamber and its anatomic structures appear normal, but drainage of the aqueous humor is impeded. In acute or chronic angle-closure glaucoma, the anterior chamber is shallow, the filtration angle is narrowed, and the iris may obstruct the trabecular meshwork at the entrance of the canal of Schlemm. Dilation of the pupil may push the root of the iris forward against the angle, and may produce pupilary block and thus precipitate an acute attack. Eyes with narrow anterior chamber angles are predisposed to acute angle-closure glaucoma attacks of various degrees of severity.

Secondary glaucoma is caused by any interference with the flow of aqueous humor from the posterior chamber into the anterior chamber and subsequently, into the canal of Schlemm. Inflammatory disease of the anterior segment may prevent aqueous escape by causing complete posterior synechia in iris bombe, and may plug the drainage channel with exudates. Other common causes are intraocular tumors, enlarged cataracts, central retinal vein occlusion, trauma to the eye, operative procedures and intraocular hemorrhage.

Considering all types together, glaucoma occurs in about 2% of all persons over the age of 40 and may be asymptotic for years before progressing to rapid loss of vision. In cases where surgery is not indicated, topical b-adrenoreceptor antagonists have traditionally been the drugs of choice for treating glaucoma.

Certain eicosanoids and their derivatives have been reported to possess ocular hypotensive activity, and have been recommended for use in glaucoma management. Eicosanoids and derivatives include numerous biologically important compounds such as prostaglandins and their derivatives. Prostaglandins can be described as derivatives of prostanoic acid which have the following structural formula:

Various types of prostaglandins are known, depending on the structure and substituents carried on the alicyclic ring of the prostanoic acid skeleton. Further classification is based on the number of unsaturated bonds in the side chain indicated by numerical subscripts after the generic type of prostaglandin [e.g. prostaglandin E₁ (PGE₁), prostaglandin E₂ (PGE₂)], and on the configuration of the substituents on the alicyclic ring indicated by α or β [e.g. prostaglandin F_(2α) (PGF_(2β))].

Prostaglandins were earlier regarded as potent ocular hypertensives, however, evidence accumulated in the last decade shows that some prostaglandins are highly effective ocular hypotensive agents, and are ideally suited for the long-term medical management of glaucoma (see, for example, Bito, L. Z. Biological Protection with Prostaglandins, Cohen, M. M., ed., Boca Raton, Fla., CRC Press Inc., 1985, pp. 231-252; and Bito, L. Z., Applied Pharmacology in the Medical Treatment of Glaucomas Drance, S. M. and Neufeld, A. H. eds., New York, Grune & Stratton, 1984, pp. 477-505. Such prostaglandins include PGF_(2α), PGF_(1α), PGE₂, and certain lipid-soluble esters, such as C₁ to C₂ alkyl esters, e.g. 1-isopropyl ester, of such compounds.

Although the precise mechanism is not yet known experimental results indicate that the prostaglandin-induced reduction in intraocular pressure results from increased uveoscleral outflow [Nilsson et.al., Invest. Ophthalmol. Vis. Sci. (suppl), 284 (1987)].

The isopropyl ester of PGF_(2α) has been shown to have significantly greater hypotensive potency than the parent compound, presumably as a result of its more effective penetration through the cornea. In 1987, this compound was described as “the most potent ocular hypotensive agent ever reported” [see, for example, Bito, L. Z., Arch. Ophthalmol. 105, 1036 (1987), and Siebold et.al., Prodrug 53 (1989)].

Whereas prostaglandins appear to be devoid of significant intraocular side effects, ocular surface (conjunctival) hyperemia and foreign-body sensation have been consistently associated with the topical ocular use of such compounds, in particular PGF_(2α) and its prodrugs, e.g., its 1-isopropyl ester, in humans. The clinical potentials of prostaglandins in the management of conditions associated with increased ocular pressure, e.g. glaucoma are greatly limited by these side effects.

In a series of co-pending United States patent applications assigned to Allergan, Inc. prostaglandin esters with increased ocular hypotensive activity accompanied with no or substantially reduced side-effects are disclosed. The U.S. Ser. No. 596,430 (filed Oct. 10, 1990, now U.S. Pat. No. 5,446,041), relates to certain 11-acyl-prostaglandins, such as 11-pivaloyl, 11-acetyl, 11-isobutyryl, 11-valeryl, and 11-isovaleryl PGF_(2α). Intraocular pressure reducing 15-acyl prostaglandins are disclosed in the application U.S. Ser. No. 175,476 (filed Dec. 29, 1993). Similarly, 11,15-9,15 and 9,11-diesters of prostaglandins, for example 11,15-dipivaloyl PGF_(2α) are known to have ocular hypotensive activity. See the patent applications U.S. Ser. No. 385,645 (filed Jul. 7, 1989, now U.S. Pat. No. 4,994,274), U.S. Ser. No. 584,370 (filed Sep. 18, 1990, now U.S. Pat. No. 5,028,624) and U.S. Ser. No. 585,284 (filed Sep. 18, 1990, now U.S. Pat. No. 5,034,413). The disclosures of all of these patent applications are hereby expressly incorporated by reference.

8-Azaprostaglandin analogs are disclosed in PCT Patent Application WO 01/46140 A1.

SUMMARY OF THE INVENTION

The present invention concerns a method of treating ocular hypertension which comprises administering to a mammal having ocular hypertension a therapeutically effective amount of a compound of formula I

wherein hatched lines represent the α configuration, a triangle represents the β configuration, a wavy line represents either the α configuration or the β configuration and a dotted line represents the presence or absence of a double bond;

D represents a covalent bond or CH₂, O, S or NH;

X is CO₂R, CONR₂, CH₂OR, P(O)(OR)₂, CONRSO₂R, SONR₂ or

Z is CH₂ or a covalent bond;

R is H or R²;

R¹ is H, R², phenyl, or COR²;

R² is C₁-C₅ lower alkyl or alkenyl and R³ is selected from the group consisting of R², phenyl, thienyl, furanyl, pyridyl, benzothienyl, benzofuranyl, naphthyl, or substituted derivatives thereof, wherein the substituents maybe selected from the group consisting of C₁-C₅ alkyl, halogen, CF₃, CN, NO₂, NR₂, CO₂R and OR.

In a still further aspect, the present invention relates to a pharmaceutical product, comprising

a container adapted to dispense its contents in a metered form; and

an ophthalmic solution therein, as hereinabove defined.

Finally, certain of the compounds represented by the above formula, disclosed below and utilized in the method of the present invention are novel and unobvious.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of 8-Azaprostaglandin analogs as ocular hypotensives. The compounds used in accordance with the present invention are encompassed by the following structural formula I:

The preferred group of the compounds of the present invention includes compounds that have the following structural formula II.

In the above formulae, the substituents and symbols are as hereinabove defined.

In the above formulae:

Preferably D represents a covalent bond or is CH₂; more preferably D is CH₂.

Preferably Z represents a covalent bond.

Preferably R is H or C₁-C₅ lower alkyl.

Preferably R¹ is H.

Preferably R³ is selected from the group consisting of phenyl and monosubstituted derivatives thereof, e.g. chloro and trifluoromethyl phenyl.

Preferably X is CO₂R and more preferably R is selected from the group consisting of H and ethyl.

The above compounds of the present invention may be prepared by methods that are known in the art or according to the working examples below. The compounds, below, are especially preferred representative, of the compounds of the present invention.

7-[2S-[3R-Hydroxy-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3R-Hydroxy-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[4-(3-Chloro-phenyl)-3R-hydroxy-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[4-(3-Chloro-phenyl)-3R-hydroxy-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3-Oxo-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3-Oxo-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[4-(3-Chloro-phenyl)-3-oxo-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[4-(3R-Chloro-phenyl)-3-oxo-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3-Oxo-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3-Oxo-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3R-Hydroxy-4-(chloro-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3R-Hydroxy-4-(chloro-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3R-Hydroxy-4-(chlorophenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3R-Hydroxy-4-(chlorophenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[4-(3-Chloro-phenyl)-3-oxo-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[4-(3-Chloro-phenyl)-3-oxo-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[4-(3-Chloro-phenyl)-3-oxo-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[4-(3-Chloro-phenyl)-3-oxo-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3-Oxo-4-(trifluoromethylphenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3-Oxo-4-(trifluoromethylphenyl)-but-1-]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3-Oxo-4-(trifluoromethylphenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3-Oxo-4-(trifluoromethylphenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3R-Hydroxy-(4-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3R-Hydroxy-(4-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3R-Hydroxy-(4-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3R-Hydroxy-(4-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester;

7-[2S-[3-Oxo-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid;

7-[2S-[3-Oxo-4-(phenyl)-but-1-enyl]-5-oxo-pyrrolidin 1-yl]-heptanoic acid;

7-[2S-[3-Oxo-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester and

7-[2S-[3-Oxo-4-(phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester.

Pharmaceutical compositions may be prepared by combining a therapeutically effective amount of at least one compound according to the present invention, or a pharmaceutically acceptable acid addition salt thereof, as an active ingredient, with conventional ophthalmically acceptable pharmaceutical excipients, and by preparation of unit dosage forms suitable for topical ocular use. The therapeutically efficient amount typically is between about 0.0001 and about 5% (w/v), preferably about 0.001 to about 1.0% (w/v) in liquid formulations.

For ophthalmic application, preferably solutions are prepared using a hysiological saline solution as a major vehicle. The pH of such ophthalmic solutions should preferably be maintained between 6.5 and 7.2 with an appropriate buffer system. The formulations may also contain conventional, pharmaceutically acceptable preservatives, stabilizers and surfactants.

Preferred preservatives that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate and phenylmercuric nitrate. A preferred surfactant is, for example, Tween 80. Likewise, various preferred vehicles may be used in the ophthalmic preparations of the present invention. These vehicles include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose and purified water.

Tonicity adjustors may be added as needed or convenient. They include, but are not limited to, salts, particularly sodium chloride, potassium chloride, mannitol and glycerin, or any other suitable ophthalmically acceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as the resulting preparation is ophthalmically acceptable. Accordingly, buffers include acetate buffers, citrate buffers, phosphate buffers and borate buffers. Acids or bases may be used to adjust the pH of these formulations as needed.

In a similar vein, an ophthalmically acceptable antioxidant for use in the present invention includes, but is not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.

Other excipient components which may be included in the ophthalmic preparations are chelating agents. The preferred chelating agent is edentate disodium, although other chelating agents may also be used in place or in conjunction with it.

The ingredients are usually used in the following amounts:

Ingredient Amount (% w/v) active ingredient about 0.001-5 preservative 0-0.10 vehicle 0-40 tonicity adjustor 1-10 buffer 0.01-10 pH adjustor q.s. pH 4.5-7.5 antioxidant as needed surfactant as needed purified water as needed to make 100%

The actual dose of the active compounds of the present invention depends on the specific compound, and on the condition to be treated; the selection of the appropriate dose is well within the knowledge of the skilled artisan.

The ophthalmic formulations of the present invention are conveniently packaged in forms suitable for metered application, such as in containers equipped with a dropper, to facilitate the application to the eye. Containers suitable for dropwise application are usually made of suitable inert, non-toxic plastic material, and generally contain between about 0.5 and about 15 ml solution.

This invention is further illustrated by the following non-limiting Examples.

EXAMPLE 1 7-[2S-[3R-Hydroxy-4-(chloro-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid EXAMPLE 1a

7-[2S-[3R-Hydroxy-4-(chloro-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid

EXAMPLE 2 7-[2S-[3R-Hydroxy-4-(chlorophenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester EXAMPLE 2a 7-[2S-[3R-Hydroxy-4-(chlorophenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester EXAMPLE 3 7-[2S-[4-(3-Chloro-phenyl)-3-oxo-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid EXAMPLE 3a 7-[2S-[4-(3-Chloro-phenyl)-3-oxo-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid EXAMPLE 4 7-[2S-[4-(3-Chloro-phenyl)-3-oxo-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester EXAMPLE 4a 7-[2S-[4-(3-Chloro-phenyl)-3-oxo-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester EXAMPLE 5 7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid EXAMPLE 5a 7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid EXAMPLE 6 7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester EXAMPLE 6a 7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester EXAMPLE 7 7-[2S-[3-oxo-4-(Trifluoromethylphenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid EXAMPLE 7a 7-[2S-[3-oxo-4-(Trifluoromethylphenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid EXAMPLE 8 7-[2S-[3-oxo-4-(Trifluoromethylphenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester EXAMPLE 8a 7-[2S-[3-oxo-4-(Trifluoromethylphenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester

The compounds of Examples 1 through 8a are made according to the methods disclosed in Examples 1 and 2 of published PCT Patent Application WO 01/46140, which is hereby incorporated by reference herein.

EXAMPLE 9 7-[2S-[3R-Hydroxy-(4-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid EXAMPLE 9a 7-[2S-[3R-Hydroxy-(4-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid EXAMPLE 10 7-[2S-[3R-Hydroxy-(4-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester EXAMPLE 10a 7-[2S-[3R-Hydroxy-(4-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester EXAMPLE 11 7-[2S-[3-oxo-4-(Phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid EXAMPLE 11a 7-[2S-[3-oxo-4-(Phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid EXAMPLE 12 7-[2S-[3-oxo-4-(Phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester EXAMPLE 12a 7-[2S-[3-oxo-4-(Phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic Acid, Ethyl Ester

The compounds of Examples 9 through 12a are made by methods analogous to the methods used to make the compounds of Examples 1 through 8, with [3-(phenyl)-2-oxo-propyl]-phosphonic acid dimethyl ester replacing [3-(3-chloro-phenyl)-2-oxo-propyl]-phosphonic acid dimethyl ester.

These compounds are tested for in vitro activity as described below and the results given in the Tables.

TABLE 1 8-Azaprostaglandin Analogs - Functional Data Example # Structure hFP hEP₁ hEP₂ hEP_(3A) hEP₄ hTP hIP hDP  3a

NA hit NA 324 54 >10⁴ NA NA  2a

NA NA NA NA 21 NA NA NA  1a

NA hit NA 324 0.02 >10⁴ NA NA  2

NA NA NA NA 65 NA NA NA  1

NA >10⁴ NA 608 0.7 >10⁴ NA NA  4a

NA NA NA NA >10⁴ NA NA NA 12a

NA NA NA NA >10⁴ NA NA NA 11a

NA NA NA hit 29 >10⁴ NA NA 12

NA NA NA NA >10⁴ NA NA NA 11

NA NA NA NA 193 NA NA NA  9

NA NA NA >10⁴ 2.4 NA NA NA 10a

NA NA NA NA 368 NA NA NA  9a

NA NA NA NA 0.9 NA NA NA 10

NA NA NA NA 1023 NA NA NA  8a

NA NA NA >10⁴ >10⁴ NA NA NA  6a

NA NA NA >10⁴ 26 NA >10⁴ NA  8

NA NA NA NA 7161 NA NA NA  7a

NA >10⁴ NA hit 86 NA NA NA  5a

NA NA NA hit 0.4 NA NA NA  7

NA >10⁴ NA hit 551 >10⁴ NA NA  6

NA NA NA NA 111 NA NA NA  5

NA NA NA hit 0.4 NA NA NA All data are EC₅₀ in nM

TABLE 2 8-Azaprostaglandin Analogs - Radioligand Binding Data Example # Structure hFP hEP₁ hEP₂ hEP_(3D) hEP₄ hTP hIP  3a

NA 300  2a

NA 300  1a

>10⁴ 0.4  2

NA 1000  1

5800 12  4a

NA >10⁴ 12a

NA >10⁴ 11a

NA 300 12

NA 8900 11

NA 1500  9

NA 18 10a

NA 600  9a

NA 9 10

 8a

NA >10⁴  6a

NA 200  8

NA >10⁴  7a

>10⁴ 500  5a

NA 5  7

NA 2200  6

NA 1200  5

NA 17 values are IC₅₀ in nM

HUMAN RECOMBINANT EP₁, EP₂, EP₃, EP₄, FP, TP, IP and DP RECEPTORS: STABLE TRANSFECTANTS

Plasmids encoding the human EP₁, EP₂, EP₃, EP₄, FP, TP, IP and DP receptors were prepared by cloning the respective coding sequences into the eukaryotic expression vector pCEP4 (Invitrogen). The pCEP4 vector contains an Epstein Barr virus (EBV) origin of replication, which permits episomal replication in primate cell lines expressing EBV nuclear antigen (EBNA-1). It also contains a hygromycin resistance gene that is used for eukaryotic selection. The cells employed for stable transfection were human embryonic kidney cells (HEK-293) that were transfected with and express the EBNA-1 protein. These HEK-293-EBNA cells (Invitrogen) were grown in medium containing Geneticin (G418) to maintain expression of the EBNA-1 protein. HEK-293 cells were grown in DMEM with 10% fetal bovine serum (FBS), 250 μg ml⁻¹ G418 (Life Technologies) and 200 μg ml⁻¹ gentamicin or penicillin/streptomycin. Selection of stable transfectants was achieved with 200 μg ml⁻¹ hygromycin, the optimal concentration being determined by previous hygromycin kill curve studies.

For transfection, the cells were grown to 50-60% confluency on 10 cm plates. The plasmid pCEP4 incorporating cDNA inserts for the respective human prostanoid receptor (20 μg) was added to 500 μl of 250 mM CaCl₂. HEPES buffered saline×2 (2×HBS, 280 mM NaCl, 20 mM HEPES acid, 1.5 mM Na₂ HPO₄, pH 7.05-7.12) was then added dropwise to a total of 500 μl, with continuous vortexing at room temperature. After 30 min, 9 ml DMEM were added to the mixture. The DNA/DMEM/calcium phosphate mixture was then added to the cells, which had been previously rinsed with 10 ml PBS. The cells were then incubated for 5 hr at 37° C. in humidified 95% air/5% CO₂. The calcium phosphate solution was then removed and the cells were treated with 10% glycerol in DMEM for 2 min. The glycerol solution was then replaced by DMEM with 10% FBS. The cells were incubated overnight and the medium was replaced by DMEM/10% FBS containing 250 μg ml⁻¹ G418 and penicillin/streptomycin. The following day hygromycin B was added to a final concentration of 200 μg ml⁻¹.

Ten days after transfection, hygromycin B resistant clones were individually selected and transferred to a separate well on a 24 well plate. At confluence each clone was transferred to one well of a 6 well plate, and then expanded in a 10 cm dish. Cells were maintained under continuous hygromycin selection until use.

RADIOLIGAND BINDING

Radioligand binding studies on plasma membrane fractions prepared for cells stably transfected with the cat or human receptor were performed as follows. Cells washed with TME buffer were scraped from the bottom of the flasks and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added as necessary to achieve a 40 ml volume in the centrifuge tubes. TME is comprised of 50 mM TRIS base, 10 mM MgCl₂, 1 mM EDTA; pH 7.4 is achieved by adding 1 N HCl. The cell homogenate was centrifuged at 19,000 rpm for 20-25 min at 4° C. using a Beckman Ti-60 or Ti-70 rotor. The pellet was then resuspended in TME buffer to provide a final protein concentration of 1 mg/ml, as determined by Bio-Rad assay. Radioligand binding assays were performed in a 100 μl or 200 μl volume.

The binding of [³H](N) PGE₂ (specific activity 165 Ci/mmol) was determined in duplicate and in at least 3 separate experiments. Incubations were for 60 min at 25° C. and were terminated by the addition of 4 ml of ice-cold 50 mM TRIS-HCl followed by rapid filtration through Whatman GF/B filters and three additional 4 ml washes in a cell harvester (Brandel). Competition studies were performed using a final concentration of 2.5 or 5 nM [³H](N) PGE₂ and non-specific binding was determined with 10⁻⁵ M unlabelled PGE₂.

For radioligand binding on the transient transfectants, plasma membrane fraction preparation was as follows. COS-7 cells were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes. The composition of TME is 100 mM TRIS base, 20 mM MgCl₂, 2M EDTA; 10N HCl is added to achieve a pH of 7.4.

The cell homogenate was centrifuged at 19000 rpm for 20 min at 4° C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding assays were performed in a 200 μl volume.

The binding of [³H] PGE₂ (specific activity 165 Ci or mmol⁻¹) at EP_(3D), receptors and [³H]-SQ29548 (specific activity 41.5 Ci mmol⁻¹) at TP receptors were determined in duplicate in at least three separate experiments. Radiolabeled PGE₂ was purchased from Amersham, radiolabeled SQ29548 was purchased from New England Nuclear. Incubations were for 60 min at 25° C. and were terminated by the addition of 4 ml of ice-cold 50 mM TRIS-HCl, followed by rapid filtration through Whatman GF/B filters and three additional 4 ml washes in a cell harvester (Brandel). Competition studies were performed using a final concentration of 2.5 or 5 nM [³H]-PGE₂, or 10 nM [³H]-SQ 29548 and non-specific binding determined with 10 μM of the respective unlabeled prostanoid. For all radioligand binding studies, the criteria for inclusion were >50% specific binding and between 500 and 1000 displaceable counts or better.

The effects of the compounds of this invention on intraocular pressure may be measured as follows. The compounds are prepared at the desired concentrations in a vehicle comprising 0.1% polysorbate 80 and 10 mM TRIS base. Dogs are treated by administering 25 μl to the ocular surface, the contralateral eye receives vehicle as a control. Intraocular pressure is measured by applanation pneumatonometry. Dog intraocular pressure is measured immediately before drug administration and at 6 hours thereafter.

The compounds of this invention are useful in lowering elevated intraocular pressure in mammals, e.g. humans.

The foregoing description details specific methods and compositions that can be employed to practice the present invention, and represents the best mode contemplated. However, it is apparent for one of ordinary skill in the art that further compounds with the desired pharmacological properties can be prepared in an analogous manner, and that the disclosed compounds can also be obtained from different starting compounds via different chemical reactions. Similarly, different pharmaceutical compositions may be prepared and used with substantially the same result. Thus, however detailed the foregoing may appear in text, it should not be construed as limiting the overall scope hereof; rather, the ambit of the present invention is to be governed only by the lawful construction of the appended claims. 

What is claimed is:
 1. A method of treating ocular hypertension or glaucoma which comprises administering to an animal having ocular hypertension or glaucoma a therapeutically effective amount of a compound represented by the general formula I;

wherein hatched lines represent the α configuration, a triangle represents the β configuration, a wavy line represents either the α configuration or the β configuration and a dotted line represents the presence or absence of a double bond; D represents a covalent bond or CH₂, O, S or NH; X is CO₂R, CONR₂, CH₂OR, P(O)(OR)₂, CONRSO₂R, SONR₂ or

Z is CH₂ or a covalent bond; R is H or R²; R¹ is H, R², phenyl, or COR²; R² is C₁-C₅ lower alkyl or alkenyl and R₃ is selected from the group consisting of R², phenyl, thienyl, furanyl, pyridyl, benzothienyl, benzofuranyl, naphthyl, or substituted derivatives thereof, wherein the substituents are selected from the group consisting of C₁-C₅ alkyl, halogen, CF₃, CN, NO₂, NR₂, CO₂R and OR.
 2. The method according to claim 1 wherein said compound is represented by the general formula II;


3. The method of claim 1 wherein Z represents a covalent bond.
 4. The method of claim 1 wherein D is CH₂.
 5. The method of claim 1 wherein X is CO₂R.
 6. The method of claim 5 wherein R is selected from the group consisting of H and ethyl.
 7. The method of claim 5 wherein R is H, or C₁-C₅ alkyl.
 8. The method of claim 1 wherein R₁ is H.
 9. The method of claim 1 wherein R³ is selected from the group consisting of phenyl, chlorophenyl and trifluoromethylphenyl.
 10. The method of claim 1 wherein said compound is selected from the group consisting of 7-[2S-[3R-Hydroxy-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3R-Hydroxy-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[4-(3-Chloro-phenyl)-3R-hydroxy-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[4-(3-Chloro-phenyl)-3R-hydroxy-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3-Oxo-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3-Oxo-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[4-(3-Chloro-phenyl)-3-oxo-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[4-(3R-Chloro-phenyl)-3-oxo-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3-Oxo-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3-Oxo-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3R-Hydroxy-4-(chloro-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3R-Hydroxy-4-(chloro-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3R-Hydroxy-4-(chlorophenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3R-Hydroxy-4-(chlorophenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[4-(3-Chloro-phenyl)-3-oxo-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[4-(3-Chloro-phenyl)-3-oxo-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[4-(3-Chloro-phenyl)-3-oxo-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[4-(3-Chloro-phenyl)-3-oxo-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3R-Hydroxy-4-(3-trifluoromethyl-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3-Oxo-4-(trifluoromethylphenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3-Oxo-4-(trifluoromethylphenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3-Oxo-4-(trifluoromethylphenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3-Oxo-4-(trifluoromethylphenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3R-Hydroxy-(4-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3R-Hydroxy-(4-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3R-Hydroxy-(4-phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3R-Hydroxy-(4-phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester; 7-[2S-[3-Oxo-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3-Oxo-4-(phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid; 7-[2S-[3-Oxo-4-(phenyl)-butyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester and 7-[2S-[3-Oxo-4-(phenyl)-but-1-enyl]-5-oxo-pyrrolidin-1-yl]-heptanoic acid, ethyl ester.
 11. An ophthalmic solution comprising a therapeutically effective amount of a compound represented by the general Formula 1

wherein hatched lines represent the α configuration, a triangle represents the β configuration, a wavy line represents the α configuration or the β configuration and a dotted line represents the presence or absence of a double bond;

D represents a covalent bond or CH₂, O, S or NH; X is CO₂R, CONR₂, CH₂OR, P(O)(OR)₂, CONRSO₂RSONR₂ Z is CH₂ or a covalent bond; R is H or R²; R¹ is H, R², phenyl, or COR²; R² is C₁-C₅ lower alkyl or alkenyl and R₃ is selected from the group consisting of R², phenyl, thienyl, furanyl, pyridyl, benzothienyl, benzofuranyl, naphthyl or substituted derivatives thereof, wherein the substituents are selected from the group consisting of C₁-C₅ alkyl, halogen, CF₃, CN, NO₂, NR₂, CO₂R and OR in admixture with a non-toxic, ophthalmically acceptable liquid vehicle, packaged in a container suitable for metered application.
 12. A pharmaceutical product, comprising a container adapted to dispense the contents of said container in metered form; and an ophthalmic solution according to claim 11 in said container. 